The present invention is related to the subject matter of U.S. Pat. No. 4,355,107 which issued Oct. 19, 1982.
Adipic acid is an important commodity in the chemical industry, particularly for consumption as a comonomer in the synthesis of polymers. Adipic acid can be obtained by oxidation of cyclohexane or cyclohexanol. Another prospective method is by the hydrogenation of muconic acid, which is a diolefinically unsaturated adipic acid derivative: ##STR1##
A potentially convenient source of muconic acid is by the microbiological oxidation of various hydrocarbon substrates. Microbiological oxidation of hydrocarbons is reviewed in Applied Microbiology, 9(5), 383(1961) and in "Advances in Enzymology", 27, 469-546(1965) by Interscience Publishers.
U.S. Pat. No. 3,383,289 describes a process for producing a methyl-substituted muconic acid and/or 2,3-dihydroxybenzoic acid which involves subjecting a C.sub.7 -C.sub.10 methylbenzene having 1-4 methyl groups and at least two consecutive unsubstituted ring carbon atoms in the presence of a nutrient medium and under fermentation conditions to the action of an orthodihydroxylating and nondecarboxylating strain of Nocardia.
The Journal of Biological Chemistry, 241(16), 3776 (1966) reports the conversion of catechol and protocatechuate to .beta.-ketoadipate by Pseudomonas putida. The conversion of catechol proceeds by the ortho pathway via a muconic acid intermediate: ##STR2## The chemical structures illustrated in the reaction scheme are catechol, muconic acid, muconolactone, .beta.-ketoadipate enollactone and .beta.-ketoadipate, respectively.
In the Journal Of Bacteriology, 134, 756(1978) there is reported a study of the ubiquity of plasmids in coding for toluene and xylene metabolism in soil bacteria. One of the mutant strains of Pseudomonas putida isolated had the ability to metabolize toluene via benzyl alcohol, benzaldehyde, benzoic acid and catechol by the ortho pathway through .beta.-ketoadipate to biomass and carbon dioxide.
The enzymes functioning in the toluene metabolism by the ortho pathway included toluene mono-oxygenase, benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, benzoate oxygenase, dihydrodihydroxybenzoate dehydrogenase, catechol 1,2-oxygenase and muconate lactonizing enzyme. The subsequently formed .beta.-ketoadipate was further assimilated to biomass and carbon dioxide. The mutant strains that metabolized toluene via the ortho pathway did not accumulate muconic acid, since the said muconic acid metabolite was further transformed in the presence of muconate lactonizing enzyme.
No known naturally occurring microorganisms (e.g., Pseudomonas putida) are known that metabolize an aromatic substrate such as toluene by the ortho pathway via muconic acid and .beta.-ketoadipate. Wild strains metabolize aromatic hydrocarbon substrates by the meta pathway via 2-hydroxymuconic semialdehyde instead of a muconic acid intermediate. Catechol 2,3-oxygenase is functional rather than catechol 1,2-oxygenase.
Thus, the potential of microbiological oxidation of an aromatic substrate such as toluene as a convenient source of muconic acid requires the construction of mutant strains of microorganisms which (1) metabolize an aromatic substrate via catechol by means of the ortho pathway, (2) allow the accumulation of muconic acid without its further assimilation, and (3) contain catechol 1,2-oxygenase which is not inhibited by accumulated muconic acid in a bioconversion medium.
Accordingly, it is an object of this invention to provide a process for construction of novel strains of microorganisms which metabolize catechol or a catechol-precursor by the ortho pathway to accumulated muconic acid.
It is another object of this invention to provide novel strains of pseudomonads which metabolize toluene or other benzoic acid-precursor to muconic acid quantitatively, with an accumulation of greater than one gram of muconic acid per liter of bioconversion medium.
Other objects and advantages of the present invention shall become apparent from the accompanying description and examples.